*Target (protein/gene name): fructose-1,6-bisphosphate aldolase *NCBI Gene # or RefSeq#:PF3D7_1444800 *Protein ID (NP or XP #) or Wolbachia#:P14223 (from http://www.uniprot.org/uniprot/P14223) *Organism:Plasmodium falciparum Etiologic Risk Group (see link below): RG2 *Background/Disease Information: P. falciparum is a protozoan parasite that is instrumental in causing malaria - the number one killer infectious disease in the world. The fructose-1,6-bisphosphate aldolase enzyme is a glycolytic enzyme which if targeted, could halt the production of ATP for the protozoan which would effectively kill it. The p. falciparum version of the enzyme also differs from the human version of the enzyme making selective targeting possible. Essentiality of this protein: Affects glycolysis so it is very essential to p. falciparum survivial (link to article: http://www.ncbi.nlm.nih.gov/pubmed/9521758) Complex of enzymes: no *EC#: 4.1.2.13 Link to BRENDA EC# page:http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.1.2.13 --Show screenshot of BRENDA enzyme mechanism schematic
From Sigma:
(1) Trizma base, 25 g for $15.40
(2) D-Fructose 1,6-Diphosphate, Tetra(cyclohexylammonium) Salt, 5 g for $102.00
(3) ß-Nicotinamide Adenine Dinucleotide, Reduced Form, Disodium Salt, 50 mg for $22.60 OR β-Nicotinamide adenine dinucleotide, reduced disodium salt, 15 vials with 5 mg per vial for $154.50
(4) a-Glycerophosphate Dehydrogenase/Triosephosphate Isomerase, 250 units at 75-200 units/mg for $48.40 Total reagent cost: $188.40 - $320.30 (depending on which reagent chosen for 3.)
Structure Available (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: Direct match-http://www.pdb.org/pdb/explore/explore.do?structureId=1A5C -- Query Coverage (if not direct match): N/A -- Max Ident (if not direct match): N/A Druggable Target (see Databases for this): yes Current Inhibitors: Expression Information (has it been expressed in bacterial cells): expressed in E. coli Purification Method: Image of protein (PyMol or etc): see below
*length of your protein in Amino Acids = 369 AA Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite = 40105.0 Daltons Molar Extinction coefficient of your protein at 280 nm wavelength:
Ext. coefficient = 40255 Abs 0.1% (=1 g/l) 1.004, assuming all pairs of Cys residues form cystines
Ext. coefficient = 39880 Abs 0.1% (=1 g/l) 0.994, assuming all Cys residues are reduced
*CDS Gene Sequence:
(Cannot seem to find gene sequence for p. falciparum)
*NCBI Gene # or RefSeq#: PF3D7_1444800
*Protein ID (NP or XP #) or Wolbachia#:P14223 (from http://www.uniprot.org/uniprot/P14223)
*Organism: Plasmodium falciparum
Etiologic Risk Group (see link below): RG2
*Background/Disease Information: P. falciparum is a protozoan parasite that is instrumental in causing malaria - the number one killer infectious disease in the world. The fructose-1,6-bisphosphate aldolase enzyme is a glycolytic enzyme which if targeted, could halt the production of ATP for the protozoan which would effectively kill it. The p. falciparum version of the enzyme also differs from the human version of the enzyme making selective targeting possible.
Essentiality of this protein: Affects glycolysis so it is very essential to p. falciparum survivial (link to article: http://www.ncbi.nlm.nih.gov/pubmed/9521758)
Complex of enzymes: no
*EC#: 4.1.2.13
Link to BRENDA EC# page: http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.1.2.13
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
-- link to paper that contains assay information: http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/General_Information/2/aldolase.Par.0001.File.tmp/aldolase.pdf
-- List cost and quantity of substrate reagents and supplier
From Sigma:
(1) Trizma base, 25 g for $15.40
(2) D-Fructose 1,6-Diphosphate, Tetra(cyclohexylammonium) Salt, 5 g for $102.00
(3) ß-Nicotinamide Adenine Dinucleotide, Reduced Form, Disodium Salt, 50 mg for $22.60 OR β-Nicotinamide adenine dinucleotide, reduced disodium salt, 15 vials with 5 mg per vial for $154.50
(4) a-Glycerophosphate Dehydrogenase/Triosephosphate Isomerase, 250 units at 75-200 units/mg for $48.40
Total reagent cost: $188.40 - $320.30 (depending on which reagent chosen for 3.)
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: Direct match-http://www.pdb.org/pdb/explore/explore.do?structureId=1A5C
-- Query Coverage (if not direct match): N/A
-- Max Ident (if not direct match): N/A
Druggable Target (see Databases for this): yes
Current Inhibitors:
Expression Information (has it been expressed in bacterial cells): expressed in E. coli
Purification Method:
Image of protein (PyMol or etc): see below
*Amino Acid Sequence:
MAHCTEYMNAPKKLPADVAEELATTAQKLVQAGKGILAADESTQTIKKRFDNIKLENTIE NRASYRDLLFGTKGLGKFISGAILFEETLFQKNEAGVPMVNLLHNENIIPGIKVDKGLVN IPCTDEEKSTQGLDGLAERCKEYYKAGARFAKWRTVLVIDTAKGKPTDLSIHETAWGLAR YASICQQNRLVPIVEPEILADGPHSIEVCAVVTQKVLSCVFKALQENGVLLEGALLKPNM VTAGYECTAKTTTQDVGFLTVRTLRRTVPPALPGVVFLSGGQSEEEASVNLNSINALGPH PWALTFSYGRALQASVLNTWQGKKENVAKAREVLLQRAEANSLATYGKYKGGAGGENAGA SLYEKKYVY
*length of your protein in Amino Acids = 369 AA
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite = 40105.0 Daltons
Molar Extinction coefficient of your protein at 280 nm wavelength:
Ext. coefficient = 40255 Abs 0.1% (=1 g/l) 1.004, assuming all pairs of Cys residues form cystines
Ext. coefficient = 39880 Abs 0.1% (=1 g/l) 0.994, assuming all Cys residues are reduced
*CDS Gene Sequence:
(Cannot seem to find gene sequence for p. falciparum)
*GC% Content for gene:
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol:
ATGGCGCACTGCACTGAGTACATGAATGCGCCGAAGAAACTGCCGGCTGACGTTGCGGAA
GAACTCGCGACCACTGCGCAAAAACTGGTTCAGGCGGGTAAAGGTATCCTGGCGGCTGAT
GAATCTACCCAAACCATCAAGAAACGCTTCGACAACATCAAGCTGGAAAACACCATCGAA
AACCGTGCGTCTTACCGCGACCTGCTGTTCGGCACCAAAGGTCTGGGTAAATTCATCTCT
GGTGCGATCCTGTTCGAGGAGACGCTGTTCCAGAAAAACGAAGCGGGCGTTCCAATGGTT
AATCTGCTGCACAACGAGAACATCATCCCGGGTATCAAAGTTGACAAAGGCCTCGTAAAT
ATCCCGTGCACCGATGAGGAAAAATCTACGCAGGGTCTCGACGGTCTGGCTGAACGTTGC
AAAGAATACTACAAAGCTGGTGCTCGCTTTGCTAAATGGCGTACCGTTCTGGTTATCGAC
ACGGCGAAGGGCAAACCGACCGACCTGAGCATCCACGAAACCGCGTGGGGCCTCGCACGT
TACGCGTCTATCTGCCAACAGAATCGTCTGGTTCCGATCGTTGAACCGGAAATTCTGGCG
GACGGCCCTCACTCTATCGAAGTTTGCGCGGTTGTTACGCAGAAGGTTCTGTCTTGCGTA
TTCAAAGCTCTGCAAGAAAATGGTGTGCTGCTGGAAGGTGCTCTGCTGAAACCGAACATG
GTTACGGCGGGTTACGAATGTACTGCGAAGACGACCACCCAGGACGTTGGTTTCCTGACC
GTTCGTACGCTGCGTCGTACGGTTCCTCCGGCGCTGCCGGGTGTTGTATTCCTGAGCGGT
GGTCAGTCTGAGGAAGAAGCATCCGTTAACCTGAATTCTATCAATGCGCTCGGTCCGCAC
CCGTGGGCGCTCACGTTCTCTTATGGTCGTGCGCTGCAGGCGTCTGTTCTGAACACCTGG
CAGGGTAAAAAAGAAAACGTGGCGAAAGCGCGTGAAGTTCTCCTGCAACGCGCTGAAGCG
AACTCTCTGGCGACCTACGGTAAGTATAAAGGCGGTGCGGGTGGTGAAAACGCTGGCGCG
TCTCTGTACGAAAAGAAGTACGTTTACTAA
*GC% Content for gene (codon optimized): 53.7%
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
link to DNA Works output text file : https://docs.google.com/file/d/0B4O2KqKh2q_-UnllMWtWRVNESmM/edit
Primer design results for 'tail' primers (this is just 2 sequences):
1 ATGGCGCACTGCACTGA 17
28 TTAGTAAACGTACTTCTTTTCGTACAGAGACGCGCCAGC 39